The central hypothesis of this proposal is that specialized regions of the host cell membrane known as glycolipid-enriched membrane (GEM) domains of lipid graphs play an important role in the biology of HIV-1 during both the entry and budding phases of the virus replication cycle. The goals are to define the role of lipid rafts in HIV replication and to analyze some of the important biological consequences. The specific aims are: 1) To extend early studies on HIV GEM domain budding to primary cells and CCR5-specific virus; 2) To determine whether GEM domains are preferred sites for HIV entry. These studies will examine the sorting of CD4 and chemokine receptors to GEM domains and whether such sorting of either molecule is necessary for virus entry; 3) To determine whether GPI anchors and palmitoylation are signals for incorporation of proteins by HIV. GPI anchors and palmitoylation have emerged as two key signals for sorting of membrane domains to GEM domains. These studies will determine whether these signals specifically target virus-encoded and host cell membrane proteins for incorporation by HIV; 4) To determine the effects of dispersion of GEM domains on HIV budding and entry. These studies will disrupt GEM domains by cholesterol depletion and inhibition of sphingolipid synthesis and analyze the effects on HIV entry and budding; 5) To determine the effect of dispersion of GEM domain inner leaflets on HIV budding and entry. These studies will use polyunsaturated fatty acids to modify the structure of GEM domain inner leaflets to determine their role in HIV budding and entry. Results of these studies should provide valuable new insights into HIV host membrane interactions and may provide the basis for novel anti-viral therapeutics.